Apparatus and method for mixing a film of fluid

ABSTRACT

A method and apparatus is provided for mixing a film of fluid, particularly a film of chemical, biochemical, or biological fluids undergoing a reaction. The apparatus comprises a means for nucleating a bubble using a discrete heat source, such as a resistor, and moving the bubble in the fluid by creating a temperature gradient, thereby mixing the fluid.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/782,542 (U.S. Pat. No. 6,513,968) which is a continuation ofU.S. patent application Ser. No. 09/137,963 (U.S. Pat. No. 6,186,659).Priority is claimed from both of the foregoing applications under 35U.S.C. 120 and those applications are incorporated herein by reference.

TECHNICAL FIELD

[0002] This invention relates to mixing fluids, and more particularlyrelates to an apparatus and method for mixing a small quantity of fluidthat is present as a film on a solid substrate.

BACKGROUND

[0003] Methods for mixing relatively large volumes of fluids usuallyutilize conventional mixing devices that mix the fluids by shaking thecontainer, by a rapid mechanical up and down motion, or by the use of arocking motion that tilts the container filled with the fluids in a backand forth motion. The conventional mixing methods normally cannot beutilized for methods involving thin films of fluid because the capillarystrength of the containment system often significantly exceeds theforces generated by shaking or rocking, thereby preventing or minimizingfluid motion in the film. The problem is illustrated by a surfacechemistry reaction where the surface is large and the available fluidsample is very small. The fluid when spread across the surface resultsin a thin film of fluid that may have a thickness of a few microns to afew millimeters. In such situations, the fluid may not adequatelycontact the entire reactive surface or the reactive compounds in thefluid may be very dilute thereby resulting in a reaction that is limitedby the rate of diffusion through the fluid. Thus, inadequate mixing canadversely affect the sensitivity or specificity of the reaction, therate of reaction, the extent of reaction, the homogeneity, or thepercent yield.

[0004] Inadequate mixing is a particular problem in chemical andbiological assays where very small samples of chemical, biochemical, orbiological fluids are typically reacted. For example, the ability toclone and synthesize nucleotide sequences has led to the development ofa number of techniques for disease diagnosis and genetic analysis.Genetic analysis, including correlation of genotypes and phenotypes,contributes to the information necessary to reveal the changes in geneswhich confer disease. New methods of diagnosis of diseases, such asAIDS, cancer, sickle cell anemia, cystic fibrosis, diabetes, musculardystrophy, and the like, rely on the detection of mutations present incertain nucleotide sequences. Many of these techniques generally involvehybridization between a target nucleotide sequence and a complementaryprobe, offering a convenient and reliable means for the isolation,identification, and analysis of nucleotides.

[0005] One typical method involves hybridization with either target orprobe nucleotide sequences immobilized on a solid support. The targetsor probes are usually immobilized on a solid support having a surfacearea of typically less than a few square centimeters. The solid supportis typically a glass or fused silica slide which has been treated tofacilitate attachment of either the targets or probes. The mobile phasecontaining reactants that react with the attached targets or probes isplaced on the support, covered with another slide, and placed in anenvironmentally controlled chamber such as an incubator. Normally, thereactants in the mobile phase diffuse through the liquid to theinterface where the complementary probes or targets are immobilized, anda reaction, such as hybridization reaction, then occurs. Preferably, themobile phase reactants are labeled with a detectable tag, such as afluorescent tag, so that the hybrid could be identified. Thehybridization reaction typically takes place over a time period that canbe many hours.

[0006] Problems are often encountered in conducting chemical orbiological assays, including use of arrays, with poor hybridizationkinetics and efficiency or reaction specificity and sensitivity, sincediffusion is the only means of moving the reactants in the mobile phaseto the interface or surface containing the immobilized reactants.Alternatively, the fluid sample must be removed from the reactionchamber, mixed in separate chambers external to the reaction chamber,and then reintroduced into the reaction chamber. Valuable fluid sampleis wasted or lost in the separate external chambers required in suchmixing process.

[0007] A method and apparatus for mixing a thin film of fluid,particularly a chemical, biochemical, or biological fluid undergoing areaction, is described in a copending application U.S. Ser. No.08/889763. The application describes a thin film of fluid between twoopposing surfaces that is mixed by moving one surface relative to theother.

[0008] The present invention describes an apparatus and method formixing of a film of fluid via nucleation of bubbles within the film. Theuse of bubbles for mixing large volumes of liquids is well known. Forexample, U.S. Pat. No. 5,443,985 to Lu et al. and U.S. Pat. No.5,605,653 to DeVos describe the mixing and aeration of large volumes ofliquid, such as a culture medium in a cell culture bioreactor byintroducing extraneous gas at the bottom of the reactor thereby creatingbubbles that travel upwards, thus mixing the liquid medium. In anothercontext, U.S. Pat. No. 5,275,787 to Yaguchi et al. describes the use ofthermal energy to generate a bubble that is then used to discharge asample liquid containing individual particles. The generation of thebubble and its use as an optical switching element for devices that haveuses in telecommunication systems and data communication systems isdescribed in U.S. Pat. No. 5,699,462 to Fouquet et al. and U.S. Pat. No.4,988,157 to Jackel et al.

SUMMARY OF THE INVENTION

[0009] The invention, in one embodiment, is an apparatus for mixing afilm of fluid, particularly a chemical, biochemical, or biological fluidwhich typically comprises a reaction mixture, the apparatus comprising afirst substrate having an inner surface and a substantially parallelsecond substrate having an inner surface that defines a closed chambertherebetween. The closed chamber is adapted to retain a quantity offluid so that the fluid is in contact with both inner surfaces. Inaddition, the apparatus comprises a means for nucleating bubbles in thefluid comprising discrete heat sources for creating individual bubblesat selected locations within the apparatus such that as each bubble isnucleated and dispelled, the fluid is displaced resulting in mixing, anda means for moving the bubbles in the fluid. The inner surface of one orboth of the substrates is functionalized with reactive moieties that canreact with the components contained in the fluid. The bubbles arenucleated by using discrete heat sources, such as resistors arranged ina predetermined pattern adjacent to the inner surface of the substrate.

[0010] In another embodiment, only the first substrate has the means fornucleating bubbles comprising discrete heat sources adjacent to theinner surface and is functionalized with reactive moieties.

[0011] The invention also provides a method for mixing a film of fluidcomprising providing a first substrate and a substantially parallelsecond substrate having inner surfaces that define a closed chambertherebetween. The chamber is adapted to retain a quantity of fluid sothat the fluid is in contact with both inner surfaces. The methodfurther comprises introducing a fluid containing a plurality ofcomponents into the closed chamber so as to provide a film of fluidtherein, nucleating a bubble within the fluid film, whereby, as eachbubble that is nucleated is dispelled, the fluid is displaced resultingin mixing, and a means for moving a bubble in the film of fluid. Thebubbles may be moved in the film of fluid by creating a temperaturegradient along which the bubbles move.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 is a schematic diagram of an apparatus of the presentinvention wherein the first substrate 10 is substantially parallel tothe second substrate 11 with a seal 15 in between.

[0013]FIG. 2 is a cross-sectional view of the apparatus shown in FIG. 1,wherein discrete resistors 13 are adjacent to the inner surface 12 ofthe first substrate 10 with a seal 15 in between the first 10 and thesecond 11 substrates.

[0014]FIG. 3 is a edge on view of an apparatus of the present invention,wherein the first substrate 10 and the second substrate 11 aresubstantially parallel with a seal 15 in between.

[0015]FIG. 3a is a cross-sectional view of the apparatus shown in FIG.3, illustrating the plurality of chemical, biochemical, or biologicalmoieties 14 attached to the inner surface 12 of the second substrate 11and discrete resistors 13 adjacent to the inner surface of the firstsubstrate 10.

DETAILED DESCRIPTION OF THE INVENTION

[0016] Overview and Definitions:

[0017] Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositions,reagents, process steps, or equipment, as such may vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

[0018] It must be noted that, as used in this specification and theappended claims, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise.

[0019] In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

[0020] The term “bubble” as used herein refers to a small ball of gas ina fluid wherein the gas originates from fluid vapor (a vapor bubble) ordissolved gas (a gas bubble) in the fluid. The word “bubble” used aloneencompasses both a gas bubble and a vapor bubble.

[0021] The term “fluid” as used herein refers to a material that canflow such as a liquid, or a semisolid.

[0022] The term “functionalization” as used herein relates tomodification of a solid substrate to provide a plurality of functionalgroups on the substrate surface. By a “functionalized surface” as usedherein is meant a substrate surface that has been modified so that aplurality of functional groups are present thereon.

[0023] The term “monomer” as used herein refers to a chemical entitythat can be covalently linked to one or more other such entities to forman oligomer. Examples of “monomers” include nucleotides, amino acids,saccharides, peptides, and the like. In general, the monomers used inconjunction with the present invention have first and second sites(e.g., C-termini and N-termini, or 5′ and 3′ sites) suitable for bindingto other like monomers by means of standard chemical reactions (e.g.,condensation, nucleophilic displacement of a leaving group, or thelike), and a diverse element which distinguishes a particular monomerfrom a different monomer of the same type (e.g., an amino acid sidechain, a nucleotide base, etc.). The initial substrate-bound monomer isgenerally used as a building-block in a multi-step synthesis procedureto form a complete ligand, such as in the synthesis of oligonucleotides,oligopeptides, and the like.

[0024] The term “oligomer” is used herein to indicate a chemical entitythat contains a plurality of monomers. As used herein, the terms“oligomer” and “polymer” are used interchangeably, as it is generally,although not necessarily, smaller “polymers” that are prepared using thefunctionalized substrates of the invention, particularly in conjunctionwith combinatorial chemistry techniques. Examples of oligomers andpolymers include polydeoxyribonucleotides, polyribonucleotides, otherpolynucleotides which are—or C-glycosides of a purine or pyrimidinebase, polypeptides, polysaccharides, and other chemical entities thatcontain repeating units of like chemical structure. In the practice ofthe instant invention, oligomers will generally comprise about 2-50monomers, preferably about 15-30 monomers.

[0025] The term “ligand” as used herein refers to a moiety that iscapable of covalently or otherwise chemically binding a compound ofinterest. Typically, when the present substrates are used in solid phasesynthesis, they are used so that “ligands” are synthesized thereon.These solid-supported ligands can then be used in screening orseparation processes, or the like, to bind a component of interest in asample. The term “ligand” in the context of the invention may or may notbe an “oligomer” as defined above. However, the term “ligand” as usedherein may also refer to a compound that is not synthesized on the novelfunctionalized substrate, but that is “pre-synthesized” or obtainedcommercially, and then attached to the substrate.

[0026] The term “sample” as used herein relates to a material or mixtureof materials, typically, although not necessarily, in fluid form,containing one or more components of interest.

[0027] The terms “nucleoside” and “nucleotide” are intended to includethose moieties which contain not only the known purine and pyrimidinebases, but also other heterocyclic bases that have been modified. Suchmodifications include methylated purines or pyrimidines, acylatedpurines or pyrimidines, or other heterocycles. In addition, the terms“nucleoside” and “nucleotide” include those moieties that contain notonly conventional ribose and deoxyribose sugars, but other sugars aswell. Modified nucleosides or nucleotides also include modifications onthe sugar moiety, e.g., wherein one or more of the hydroxyl groups arereplaced with halogen atoms or aliphatic groups, or are functionalizedas ethers, amines, or the like. As used herein, the term “amino acid” isintended to include not only the L-, D- and nonchiral forms of naturallyoccurring amino acids (alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine), but also modified amino acids, amino acidanalogs, and other chemical compounds which can be incorporated inconventional oligopeptide synthesis, e.g., 4-nitrophenylalanine,isoglutamic acid, isoglutamine, ε-nicotinoyl-lysine, isonipecotic acid,tetrahydroisoquinoleic acid, α-aminoisobutyric acid, sarcosine,citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, 4-aminobutyric acid, and the like.

[0028] Accordingly, the invention in a first embodiment is directed toan apparatus, shown in FIGS. 1 and 2, comprising a first substrate 10having an inner surface 12 that can retain a film of fluid and a meansof nucleating bubbles in the film of fluid. The substrate may becomposed of any material that is compatible with the fluids with whichthe surface comes in contact with, such as, for example, glass, silicon,fused silica, metal, ceramic, plastic, or other polymers such as nylon.The substrate may be rigid or flexible, and may define a shape that issubstantially planar in the shape of a circle, an ellipse, a square, arectangle, a triangle, or any other convenient substantially planarshape. The surface area of the inner surface that comes in contact withthe fluid is on the order of about 10 mm² to about 20 cm², morepreferably from about 100 mm² to about 10 cm², and most preferably about200 mm² to about 10 cm². The volume of the fluid that can be retained bythe inner surface is less than about 300 μl, more preferably less thanabout 150 μl, even more preferably less than about 50 μl.

[0029] The inner surface of the substrate has means for nucleatingbubbles in the film of fluid, comprising discrete heat sources,radiofrequency sources, microwave sources, light sources, or mechanicalsources. In the preferred embodiment, the means for nucleating bubblescomprise discrete heat sources, preferably resistors 13. The resistorsare adjacent to the inner surface of the substrate and in thermalcommunication with the film of fluid retained thereon. The resistors areelectrically connected to a control circuit that controls the voltagesor currents applied to the resistors to nucleate the bubble. Bubbles arethus nucleated by heating the fluid in the local environment around theresistors. Subsequently, the control circuit may selectively reduce theelectrical input to the resistors to allow the bubble to dissipate orcollapse.

[0030] The apparatus for mixing a film of fluids also has means formoving a bubble through the fluid. In the preferred embodiment, as aheat source is turned off, a neighboring heat source may be turned on,thereby creating a temperature gradient in the fluid. The bubble willmove along the temperature gradient from the region of lower temperaturetowards the region of higher temperature. The movement of the bubblealong the temperature gradient, caused by thermocapillary action,displaces the local fluid thereby mixing it. Thus, the control circuitcan control the temperature gradient. In addition, the control circuitcan control the ambient temperature of the film of fluid by supplying anidentical amount of voltage or current to nearly all the resistors atthe same time.

[0031] The fluid can be mixed locally by moving a bubble back and forthbetween two neighboring heat sources. Alternatively, a plurality ofbubbles can be nucleated at a plurality of locations and movedindependently of one another. In addition, a pre-fabricated pattern ofheat sources can be created such that the bubble is moved from one heatsource to the next along the temperature gradient dictated by thepattern. The pattern of the heat sources can be linear, circular,rectangular, or any other convenient pattern. Moving the bubble along acircular pattern, for example, induces a stirring motion in the film offluid. Similarly, if the temperature gradient is linear, the bubble willmove back and forth along the line thereby inducing a back and forthmotion in the fluid.

[0032] The inner surface of the substrate is functionalized withreactive moieties to provide a plurality of functional groups on thesubstrate surface, typically hydroxyl, carboxylic, amido, or aminogroups, that are preferably bound to a ligand. The reactive moieties canbe covalently bound or otherwise chemically bound to the ligand througha variety of methods known in the art. Optimally, the density of thechemical, biochemical, or biological ligands is greater than about 100per square micron, more preferably greater than about 1,000 per squaremicron, an even more preferably greater than about 5,000 per squaremicron. The ligands are selected from a monomeric species having atleast one reactive site, DNA, RNA, proteins, peptides, prions, reagents,and combinations, derivatives, and modifications thereof. See forexample, Kreiner (1996) “Rapid Genetic Sequence Analysis Using a DNAProbe Array System,” American Laboratory March: 39-43; Lipshutz et al.(1995) “Using Oligonucleotide Probe Arrays to Access Genetic Diversity,”BioTechniques 19: 442-447; Fodor et al. (1991) “Light-directed,Spatially Addressable Parallel Chemical Synthesis,” Science 252:767-772; Medlin (1995) “The Amazing Shrinking Laboratory,” EnvironmentalHealth Perspectives 103: 244-246; Southern et al. (1992) “Analyzing andcomparing Nucleic Acid Sequences by Hybridization to Arrays ofOligonucleotides: Evaluation Using Experimental Models,” Genomics 13:1008-1017; and Gacia et al. (1996) “Detection of Heterozygous Mutationsin BRCA1 using High Density Oligonucleotide Arrays and Two-colorFluorescence Analysis,” Nature Genetics 14: 441-447; and U.S. Pat. Nos.5,585,639; 5,601,980; and 5,551,487.

[0033] Optionally, a seal 15 can be attached to the outer periphery ofthe substrate, thus creating a chamber for the fluid with a definedthickness. The fluid is applied to an application site near the centerof the apparatus, and then distributed across the first surface to givethe film of fluid. Prior to the initiation of the mixing process, thefluid can be distributed across the inner surfaces of the substrate by,for example, rotating the apparatus, or by spreading with a cover slip.In addition, a second substrate 11, or a simple glass cover slip isplaced on top of the seal where the substantially parallel substratesdefine a closed chamber 16. The closed chamber may be a micron toseveral millimeters in thickness, preferably from about 5 microns toabout 100 microns in thickness.

[0034] Alternatively, two or more different fluids, each containing areactive moiety, can be introduced into the closed chamber and mixed tosubstantial homogeneity. This is advantageous when it is inconvenient ornot possible to attach the ligands to the reactive moiety on the innersurface of the solid substrate.

[0035] In addition, a dye, label, tag, reagent, or derivatives,modifications, or combinations thereof may be dried, lyophilized, orotherwise attached to the inner surface of the substrate. The chemical,biochemical, or biological fluid is introduced into the closed chamber,and the mixing process initiated to allow the desired reaction orlabeling to occur. The reaction can be monitored after the mixingprocess is completed or interspersed with the mixing process ifnecessary or desired. This process is particularly advantageous forfluids or reactions which are optically dense and, therefore, must beanalyzed in a very thin section or film by a spectrophotometer or otheranalytical means. The process is also an advantage for specimens such ascellular suspensions or thin biopsies which will be visually inspectedin thin sections or films for accurate count, diagnosis, or otheranalysis. This process is also an advantage when the sample isparticularly valuable or only available in minute quantities.

[0036] Electrical connection (not shown) is made to the resistorsadjacent the inner surface of the substrate, and a bubble is nucleatedat one or more resistors. Turning off a resistor removes the heat andcollapses the nucleated bubble, thereby displacing the liquid resultingin mixing. Preferably, as one resistor is turned off, if a neighboringresistor is activated, a temperature gradient is created and thenucleated bubble moves towards the hotter area resulting in a mixingaction. The mixing action is continued for the required period of time.

[0037] The apparatus having closed edges, shown in FIG. 3, mayadditionally include a seal 15 between the two opposing inner surfaces.The seal can be solid or flexible, and is fabricated from, for example,adhesives, rubber, plastic, glass, or metal. The apparatus preferablyincludes an opening in one of the substrates or in the seal forintroducing fluid into the closed chamber. The opening may be a port orother entrance. The fluid may be introduced by centrifugal means,pressure means, vacuum means, positive displacement means, or othermeans known in the art.

[0038] The inner surface of first solid substrate 10 can have both themeans for nucleating bubbles in the film of fluid and be functionalizedwith reactive moieties. Alternatively, as shown in FIG. 3a, the innersurface of one of the substrates can have the means for nucleatingbubbles 13 in the film of fluid and the inner surface of the othersubstrate can be functionalized with reactive moieties 14, wherein thefluid is retained in the closed chamber 16. Finally, the inner surfacesof both substrates can have means for nucleating bubbles in the film offluid and both can be functionalized with reactive moieties that arebound to ligands which are identical or preferably different.

[0039] The apparatus can be adapted for automated use, such as throughthe use of various controllers, computers, and the like. In addition,the apparatus can be adapted for use with multiple fluid chamberssimultaneously, where the temperature controlled environment is capableof containing and mixing multiple fluid chambers.

EXAMPLES Example 1 Hybridization to DNA arrays

[0040] An array of DNA probes is constructed by attaching a plurality ofknown probes comprising oligomers, PCR product, or cDNA at specificlocations on the inner surface of the substrate using techniques wellknown in the art. The substrate is a circular glass slide having asurface area of about 100 mm², and having resistors on the inner surfacethat are connected to a electrical source. A rubber seal is placedaround the outer edge of the substrate. About 50 μl of reactive fluid,comprising a sample of mRNA, is placed on the inner surface of the solidsubstrate. A glass slide is placed parallel to the first substrate andon top of the seal. The substrate is then rotated by a rotatingmechanism, such as a motor, at an appropriate rotational speed in orderto distribute the reactive fluid across the first surface of thesubstrate. The rotational speed may, for example, be in the range of afew hundred rpm to several thousand rpm. Once the fluid is distributedover the surface of the substrate, the fluid may be heated to an optimumtemperature for the reaction. For example, the fluid may be heated to65° C. for hybridization studies where the surface probes are cDNA, orthe fluid may be heated to 37° C. where the surface probes areoligomers.

[0041] For mixing the fluid, voltage or current is applied to at leastone of the resistors that nucleates a bubble in the fluid. Then, voltageor current is turned off while voltage or current to the neighboringresistor is turned on. This creates a temperature gradient and thebubble is lured towards the hotter zone. The bubble is thus moved in acircular pattern, inducing a stirring motion in the film of fluid. Afterhybridization is complete, the sample is removed from the apparatus, theinner surface of the substrate is washed, and the apparatus is thenanalyzed to determine the quantity of mRNA that has hybridized to eachlocation. The entire process can be automated by use of computercontrol.

Example 2 Hybridization to RNA arrays

[0042] An array of mRNA probes in a rectangular pattern is constructedat specific locations on the inner surface of the solid substrate usingtechniques well known in the art. The substrate is a rectangular glassslide having a surface area of about 100 mm². A second substrate ofsilicon has heating sources that are resistors adjacent to the innersurface that are connected to a electrical source. The resistors arearranged in a square pattern. The two inner surfaces are held inparallel to each other in an opposing relationship, and then a glassseal is applied between the two substrates. Into the closed chamber thuscreated, about 50 μl of reactive fluid, comprising a sample of DNA, isplaced by positive pressure. The fluid is then heated to 45° C. byapplying a constant predetermined voltage to all the electrodes.

[0043] As in Example 1, additional voltage or current is applied to atleast one of the resistors that nucleates a bubble in the fluid. Then,voltage or current is returned to the base level while voltage orcurrent to the neighboring resistor is increased. This creates atemperature gradient and the bubble is moved in a square pattern,inducing a stirring motion in the film of fluid. After hybridization iscomplete, the sample is removed from the apparatus, the inner surface ofthe substrate is washed, and the apparatus is then analyzed to determinethe quantity of DNA hybridized at each location.

Example 3 Solid Phase Synthesis of Polystyrene

[0044] Styrene is covalently bound via the aromatic ring to specificlocations on the inner surface of the first substrate using techniqueswell known in the art, such that the ethylene moiety remains reactive.The first substrate is a circular silicon slide having a surface area ofabout 10 cm², and having a plurality of resistors on the inner surfacethat are connected to a electrical source The resistors are arranged incircular pattern. About 250 μl of reactive fluid, comprising a sample ofstyrene and 2,2′-azo-bis-isobutyronitrile as the free radical initiator,is placed on the inner surface of the first solid substrate. The innersurface of a second substrate is held parallel to the inner surface ofthe first substrate, and a glass seal is then applied between the twosubstrates. The apparatus is rotated by a rotating mechanism, as inExample 1, to evenly distribute the fluid. The fluid is then heated to30° C. as in Example 2.

[0045] Additional voltage or current is applied to at least one of theresistors that nucleates a bubble in the fluid. Then, voltage or currentis returned to the base level while voltage or current to theneighboring resistor is applied. This creates a temperature gradient andthe bubble is moved in a back and forth pattern across the length of thesolid substrate. After reaction is complete, the sample is removed fromthe apparatus, the inner surface of the first substrate is washed, and afresh sample of fluid containing monomer suitable for polymerization isadded. The steps above are repeated until the desired length of thepolymer is obtained. The polymer chain is then dissociated from thefirst substrate to give free polystyrene.

[0046] It is to be understood that while the invention has beendescribed in conjunction with preferred specific embodiments thereof,the foregoing description, as well as the examples which follow, areintended to illustrate and not limit the scope of the invention. Otheraspects, advantages and modifications within the scope of the inventionwill be apparent to those skilled in the art to which the inventionpertains.

1. A method comprising: providing first and second subtrates and a seal;placing the second substrate on top of the seal and first substrate soas to define a closed chamber therebetween, said chamber adapted toretain a quantity of fluid so that the fluid is in contact with bothinner surfaces, and wherein at least one of said inner surfaces isfunctionalized with polynucleotides, polypeptides, or polysaccharides;and introducing a fluid containing a plurality of components into theclosed chamber so as to provide a quantity of fluid therein in contactwith both inner surfaces.
 2. A method according to claim 1 wherein thepolynucleotide is a polyribonucleotide.
 3. A method according to claim 1wherein the chamber is adapted to retain a film of fluid in contact withboth inner surfaces.
 4. A method according to claim 3 wherein thechamber is up to several millimeters in thickness.
 5. A method accordingto claim 4 wherein the inner surfaces of the first and second substratesare substantially parallel.
 6. A method according to claim 4 wherein thechamber is up to three millimeters in thickness.
 7. A method accordingto claim 6 wherein the chamber is up to two millimeters in thickness. 8.A method according to claim 1 wherein the fluid is introduced into theclosed chamber by first introducing the fluid onto the first substratecarrying the seal then placing the second substrate on top of the seal.9. A method according to claim 1 wherein the seal is attached to thefirst substrate.
 10. A method according to claim 1 wherein the innersurfaces of the first substrate is functionalized with thepolynucleotides, polypeptides, or the polysaccharides.
 11. A methodaccording to claim 1 wherein the inner surface of the second substrateis functionalized with the polynucleotides, polypeptides, or thepolysaccharides.
 12. A method according to claim 1 wherein the innersurface of the first and second substrate is each functionalized withpolynucleotides, polypeptides, or the polysaccharides.
 13. A methodaccording to claim 1 wherein one of the substrates has an opening forintroducing fluid into the closed chamber.
 14. A method according toclaim 1 wherein the seal has an opening for introducing fluid into theclosed chamber.
 15. The method of claim 1 wherein the at least one ofthe inner surfaces is functionalized with polynucleotides.
 16. Themethod of 1 wherein the at least one of the inner surfaces isfunctionalized with polypeptides.
 17. An apparatus comprising first andsecond subtrates and a seal, wherein the second substrate is positionedon top of the seal and first substrate so as to define a closed chambertherebetween, said chamber retaining a quantity of fluid therein so thatthe fluid is in contact with both inner surfaces, and wherein at leastone of said inner surfaces is functionalized with polynucleotides,polypeptides, or polysaccharides.
 18. An apparatus method according toclaim 17 wherein the chamber is up to several millimeters in thickness.19. An apparatus according to claim 18 wherein the inner surfaces of thefirst and second substrates are substantially parallel.
 20. An apparatusaccording to claim 18 wherein the chamber is up to three millimeters inthickness.
 21. An apparatus according to claim 17 wherein the innersurface of the first and second substrate is each functionalized withpolynucleotides, polypeptides, or the polysaccharides.
 22. An apparatusaccording to claim 17 wherein one of the substrates has an opening forintroducing fluid into the closed chamber.
 23. An apparatus according toclaim 17 wherein the seal has an opening for introducing fluid into theclosed chamber.